Universal autofocus for quantitative volumetric microscopy of whole mouse brains

Abstract

Unbiased quantitative analysis of macroscopic biological samples demands fast imaging systems capable of maintaining high resolution across large volumes. Here we introduce RAPID (rapid autofocusing via pupil-split image phase detection), a real-time autofocus method applicable in every widefield-based microscope. RAPID-enabled light-sheet microscopy reliably reconstructs intact, cleared mouse brains with subcellular resolution, and allowed us to characterize the three-dimensional (3D) spatial clustering of somatostatin-positive neurons in the whole encephalon, including densely labeled areas. Furthermore, it enabled 3D morphological analysis of microglia across the entire brain. Beyond light-sheet microscopy, we demonstrate that RAPID maintains high image quality in various settings, from in vivo fluorescence imaging to 3D tracking of fast-moving organisms. RAPID thus provides a flexible autofocus solution that is suitable for traditional automated microscopy tasks as well as for quantitative analysis of large biological specimens.

Modeling a 3-D multiscale blood-flow and heat-transfer framework for realistic vascular systems

“…Many levels of vasculature would be required to achieve this using RRT method, which is tantamount to recreating the entire capillary blood network as obtained from Refs. 1 , 2 , 54 . A model that can simulate blood flow and heat transfer needs to map pressure boundary conditions across the vascular network.…”

“…Technological advancements are enabling visualization and modeling of the vasculature 1 , 2 with ever-increasing resolution, providing highly detailed blood vessel domains. These domains can be further used to simulate many bio-physical mechanisms.…”

BRAin-wide Neuron quantification Toolkit reveals strong sexual dimorphism in the evolution of fear memory

“…Here, we presented BRANT, a combination of high-resolution LSM and scalable computational analysis. On the optics side, the use of RAPID autofocusing guarantees high contrast and resolution across the entire sample (Silvestri et al 2021) are specific to those neuronal regions, which in turn makes it difficult to form an overview when integrating the data together. Indeed, small changes of experimental setting (from the behavioural task to the age and sex of animals) can lead to contrasting outcomes, making it difficult to obtain a comprehensive view of brain circuits underlying memory formation, consolidation and retrieval.…”

Comparison of Different Tissue Clearing Methods for Three-Dimensional Reconstruction of Human Brain Cellular Anatomy Using Advanced Imaging Techniques

“…The clearing methods employed in this study were developed mainly for animal models application, in particular mouse brain clearing. The possibility of coupling whole-brain clearing with fast imaging with LSFM has opened the possibility to study not only the cytoarchitecture of the brain ( Silvestri et al, 2021 ), but also investigating neuronal activation using transgenic animals expressing immediate early genes ( Franceschini et al, 2020 ). Many applications ( Ueda et al, 2020b ; Weiss et al, 2021 ) have promoted the field of tissue clearing, prompting scientists to obtain higher transparency and labeling.…”

“…This can be reached with classical histological techniques using thin slices ≤100 μm, highlighting issues related to the 3D reconstruction of extended parts of tissue up to the reconstruction of the whole brain ( Amunts et al, 2013 ; Ding et al, 2015 ). In more recent years, major advances in clearing techniques ( Costantini et al, 2019 ) and the advent of fast light-sheet fluorescence microscopy (LSFM) systems ( Power and Huisken, 2017 ; Olarte et al, 2018 ; Hillman et al, 2019 ; Ueda et al, 2020a ; Silvestri et al, 2021 ) have allowed the achievement of rapid 3D histology of whole organs up to imaging transparent rodent bodies ( Pan et al, 2016 ; Kubota et al, 2017 ; Cai et al, 2019 ). However, human tissue transparency is extremely challenging due to the autofluorescence contributions in aged tissue ( Ueda et al, 2020a , b ; Pesce et al, 2021a ).…”